We have already identified the T4 DNA polymerase as a major determinant in the frequency and specificity of spontaneous frameshift mutation, through the genetic analysis of mutation in the T4 rII genes. Our recent work has extended this analysis to the level of DNA sequencing of frameshift mutations. This more refined analysis confirms the general conclusions of the genetic analysis, but more importantly begins to identify the precise nature of the specificity changes promoted by mutant DNA polymerases. These specificity changes are of more than one kind, suggesting that several mechanisms of frameshift mutagenesis contribute to spontaneous frameshift mutation. Two novel classes of frameshift mutations resulting from previously undescribed misaligned DNA intermediates have been identified. Moreover, there are initial indications that primary DNA sequences may be important factors in defining frameshift mutation in the polymerase mutant tsL141. Construction of novel M13-T4rII hybrids has been completed. These hybrids should permit more efficient "genetic" cloning of T4 frameshift sequences, the ready genetic analysis of frameshift mutations produced in vitro, and the development of new DNA substrates for further probing specific mechanisms of frameshift and deletion mutations.